1. Field of the Invention
The invention relates to pourable animal feed additives based on fermentation liquor and containing D-pantothenic acid and/or a salt thereof, and to a process for the preparation of such additives.
2. Discussion of the Background
Pantothenic acid is produced worldwide on a scale of several thousand tons per year, and demand for the product is increasing. Much of the pantothenic acid that is produced is used for feeding productive livestock such as poultry and pigs.
Pantothenic acid can be produced by biotechnology or by chemical synthesis. Biotechnology methods involve fermentation of suitable microorganisms in appropriate nutrient solutions. In chemical synthesis, DL-pantolactone is an important precursor. It is prepared in a multi-step process from formaldehyde, isobutylaldehyde and cyanide where the racemic mixture is resolved, the D-pantolactone is condensed with xcex2-alanine, and D-panthothenic acid is thus obtained.
The typical commercial form of pantothenic acid is the calcium salt of D-pantothenic acid. The calcium salt of the racemic mixture of D,L-pantothenic acid is also common.
The advantage of preparing pantothenic acid by means of fermentation of microorganisms is that the desired stereoisomeric form, namely the D form, which is free of L-pantothenic acid, is formed directly.
Various types of bacteria, such as, for example, Escherichia coli, Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes, and also yeasts, such as, for example, Debaromyces castellii, are able to produce D-pantothenic acid under suitable fermentation conditions, as is shown in EP-A-0 493 060, EP-A-0 590 857 and WO 97/10340. Especially suitable microorganisms are the Escherichia coli IFO3547 derivatives described therein, such as, for example, the strains FV5069/pFV31 or FV5069/pFV202.
In the preparation of D-pantothenic acid by fermentation, as is described in EP-A-0 493 060, EP-A-0 590 857 and WO 97/10340, a microorganism capable of producing D-panthothenic acid is cultivated in a suitable nutrient medium and the D-pantothenic acid that forms is then isolated, purified and prepared in the form of the calcium salt in a complicated manner.
Suitable nutrient media contain a carbon source, a nitrogen source, a phosphorous source, salts, trace elements and vitamins, and optionally, complex media additives, such as yeast extract. Examples of the carbon source include glucose, starch flour hydrolysate, sucrose and molasses. An example of the nitrogen source is ammonium sulfate. An example of the phosphorous source is potassium phosphate.
According to the current prior art described in WO96/33283 and EP-A-0 590857, the calcium salt of D-pantothenic acid is obtained from the pantothenic acid-containing fermentation liquor by means of a complex isolation and purification operation. After first separating off the biomass by filtration or centrifugation, the filtrate is further purified by means of activated carbon or by column chromatography. After reaction of the resulting solutions with calcium hydroxide, the desired Ca salt is allowed to crystallize.
According to WO 96/33283, the filtrate is decoloured with activated carbon in the first column. A pH value of 3.0 is adjusted using concentrated hydrochloric acid, and the liquid is then purified continuously over two further columns packed with activated carbon. Elution of the D-pantothenic acid takes place with the aid of methyl alcohol. Subsequent neutralisation using Ca(OH)2 powder yields a solution from which calcium D-pantothenate is obtained by crystallisation at 5xc2x0 C.
In the method described in EP-A-0 590 857, the filtrate is first purified with the aid of cation- and anion-exchanger columns. Elution takes place with hydrochloric acid. The eluted fraction is then neutralised using Ca(OH)2; activated carbon is added thereto and the whole is filtered off. The filtrate that is obtained is then extracted in a low molecular weight alcohol (methanol, ethanol, isopropanol), and calcium D-pantothenate is obtained by crystallisation.
The calcium D-pantothenate prepared in the described manner is used as an additive in animal feed.
According to the prior art, salts of D-pantothenic acid or D,L-pantothenic acid are obtained by chemical synthesis or from fermentation liquors and then added in pure form to feeds.
The object of the invention is to provide more readily processable forms of D-panthothenic acid and its salts and processes for the preparation thereof for feeds.
The present invention provides a pourable animal feed additive based on fermentation liquor and containing D-pantothenic acid and/or salts thereof. The feed additive is characterized in that
a) it contains the biomass formed during the fermentation in an amount of from xe2x89xa70 to 100%; and
b) it contains at least the predominant part of the further constituents of the fermentation liquor; and
c) it is in solid form, in a particle size distribution of from 20 to 2000 xcexcm, especially from 50 to 800 xcexcm, more especially from 150 to 600 xcexcm, and is pourable.
In a preferred embodiment of the invention, the pourable animal feed additive containing D-pantothenic acid and/or salts thereof is characterized in that it additionally contains, in solid form, an amount of chloride-containing constituents in a concentration of  less than 3 mg per g of additive, preferably  less than 2 mg per g of additive and especially  less than 1.5 mg per g of additive.
The additives are generally compacted, granulated, or fine-grained, but in any case, pourable form, according to requirements, and contain varying amounts of biomass. The apparent density is from 200 to 800 kg/m3, especially approximately from 400 to 700 kg/m3.
Pourable in the context of this invention means freely flowing non-clumped particles of a predetermined size which are able to be dispensed from a container. The additives are readily pourable-and storage stable.
If the biomass is separated off further, inorganic solids, for example those which have been added during the fermentation, are generally removed. In addition, the additive according to the invention contains at least the predominant part of the further substances, especially organic, that have been formed or added and are dissolved in the fermentation liquor, insofar as they have not been separated off by suitable processes.
Such substances include organic by-products that are produced and secreted in addition to D-pantothenic acid by the microorganisms used in the fermentation. They include L-amino acids, selected from the group L-methionine, L-lysine, L-valine, L-threonine, L-alanine or L-tryptophan, especially L-valine. They also include organic acids carrying from one to three carboxyl groups, such as, for example, acetic acid, lactic acid, citric acid, malic acid or fumaric acid. Finally, they also include sugars, such as, for example, trehalose. Such compounds may be desirable, in that they improve the nutritional value of the additive.
In a preferred form there is prepared a fermentation liquor containing D-pantothenic acid and/or salts thereof, wherein
a) the fermentation takes place in a substantially chloride-free medium,
b) the resulting fermentation liquor, optionally after separation of the biomass and concentration, is dried, compacted, spray dried, spray granulated or granulated or applied to a carrier or embedded in a stabilising matrix.
The fermentation liquors which are suitable for the process according to the invention are obtained using microorganisms suitable for the production of D-pantothenic acid and that contain D-pantothenic acid and/or salts thereof. The salts are generally sodium, potassium, ammonium, magnesium or calcium salt.
The microorganisms may be fungi or yeasts, such as, for example, Debaromyces castellii, or Gram-positive bacteria, for example of the genus Corynebacterium, or Gram-negative bacteria, such as, for example, those of the family Enterobacteriaceae. In the case of the family of the Enterobacteriaceae, special mention may be made of the genus Escherichia and of the species Escherichia coli. Within the species Escherichia coli, mention may be made of the so-called K-12 strains, such as, for example, strains MG1655 or W3110 (Neidhard et al.: Escherichia coli and Salmonella. Cellular and Molecular Biology (ASM Press, Washington D.C.)), or of the Escherichia coli wild type strain IFO3547 (Institut fxc3xcr Fermentation, Osaka, Japan) and mutants derived therefrom both of which are incorporated herein by reference. Of the strains produced from IFO3547, FV5069/pFV31 (EP-A-0 590 857) and FV5069/pFV202 (WO 97/10340) are prominent. In the case of the genus Corynebacterium, special mention may be made of the species Corynebacterium glutamicum. 
The above-described microorganisms can be cultivated for the purposes of D-panthothenic acid production continuously or discontinuously by the batch or fed batch or repeated fed batch process. A summary of known cultivation methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einfxc3xchrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994) both of which are incorporated herein by reference.
The culture medium to be used must meet the requirements of the microorganisms to be used in a suitable manner. The fermentation medium is substantially free of chloride-containing constituents. According to the invention, the concentration of chloride ions in the production fermenter is  less than 300 mg/l, preferably  less than 200 mg/l and very especially preferably  less than 150 mg/l. There may be used as the carbon source sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as soybean oil, sunflower oil, groundnut oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. Those substances may be used individually or in the form of a mixture. There may be used as the nitrogen source organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or in the form of a mixture. There may be used as the phosphorus source potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium must also contain salts of metals such as magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, may be used in addition to the above-mentioned substances. Precursors of D-pantothenic acid, such as aspartate, xcex2-alanine, ketoisovalerate, ketopantoic acid or pantoic acid, and, optionally, salts thereof, may also be added to the culture medium. The mentioned substances may be added to the culture in the form of a single batch, or they may be fed in a suitable manner during the cultivation.
In order to control the pH value, ammonia or ammonia water or other basic compounds, such as sodium hydroxide, potassium hydroxide or calcium hydroxide are used. If acid compounds are required to control the pH value, phosphoric acid or sulfuric acid may expediently be used. In order to prepare the calcium salt of pantothenic acid directly, calcium hydroxide in the form of an aqueous suspension is used during the fermentation. In order to control the development of foam, anti-foams such as fatty acid polyglycol esters, may be used. In order to maintain the stability of plasmids, suitable substances having a selective action, for example antibiotics, are optionally added to the medium. In order to maintain aerobic conditions, oxygen or gas mixtures containing oxygen such as air, are introduced into the culture. The temperature of the culture is normally from 20xc2x0 C. to 45xc2x0 C. and preferably from 25xc2x0 C. to 40xc2x0 C. The culture is continued until the maximum amount of D-pantothenic acid has formed, which is generally within a period of 10 hours to 160 hours.
The fermentation liquors thus obtained usually have a content of dry matter of 7.5 to 25 wt. % and contain from 2 to 20 wt. % D-pantothenic acid. Fermentation processes in which D-pantothenic acid is present in the dry matter in an amount of at least 20 wt. % when the fermentation is complete are especially advantageous. It is also advantageous for the amount of sugar in the fermentation to be limited at least at the end of the fermentation, but advantageously for at least 30% of the duration of the fermentation. That means that the concentration of usable sugar in the fermentation medium is maintained at, or reduced to from xe2x89xa70 to 3 g/l during that time.
For the preparation of the additives according to the invention, the fermentation liquors containing D-pantothenic acid and/or salts thereof are preferably first freed of all or part of the biomass by known separation methods such as centrifugation, filtration, decantation or a combination thereof. However, it is also possible according to the invention to leave all of the biomass in the fermentation liquor. The suspension obtained in that manner is then concentrated preferably to not more than 60 wt. % dry matter and worked up to a powder, for example with the aid of a spray drier or a lyophilising apparatus. The powder is then converted into a coarser-grained, readily pourable, storable and largely dust-free product by suitable compacting or granulating processes. In the granulating or compacting operation it is advantageous to use conventional organic or inorganic auxiliary substances, or carriers, such as starch, gelatin, cellulose derivatives or similar substances, as are conventionally employed as binders, gelling agents or thickeners in the processing of foodstuffs or feeds, or further substances such as, for example, silicas, silicates or stearates.
Alternatively, the product may be applied to an organic or inorganic carrier substance that is known and conventionally employed in the processing of feeds, such as, for example, silicas, silicates, meals, brans, flours, starches, sugars or the like, and stabilised by means of conventional thickeners or binders. Relevant examples and processes are described in the literature (Die Mxc3xchle+Mischfuttertechnik 132 (1995) 49, page 817) which is incorporated herein by reference.
The novel solid products according to the invention that contain D-pantothenic acid and/or salts thereof and that can be prepared by the above-described process contain from 20 to 80 wt. %, preferably from 30 to 75 wt. %, D-pantothenic acid. They generally contain inorganic constituents in an amount of from 2.5 to 25 wt. % and optionally organic by-products in an amount of from  greater than 0 to 30 wt. %. The content of dry biomass is from xe2x89xa70 to 35 wt. %. The water content is preferably  less than 5 wt. %.
The novel products according to the invention that contain D-pantothenic acid and/or salts thereof and that are prepared by the above-described process are distinguished by a particle size distribution of from 20 xcexcm to 2000 xcexcm, preferably from 50 xcexcm to 800 xcexcm and especially from 150 xcexcm to 600 xcexcm. The content of very fine dust ( less than 10 xcexcm) is approximately from 0 wt. % to 10 wt. %, preferably approximately from 0 wt. % to 5 wt. %. The product is used as a feed additive.
The concentration of D-pantothenic acid can be determined by known methods (Velisek; Chromatographic Science 60, 515-560 (1992)).
The particle size distribution can be determined by methods of laser diffraction spectrometry. Corresponding methods are described in the textbook on xe2x80x9cTeilchengrxc3x6ssenmessung in der Laborpraxisxe2x80x9d by R. H. Mxc3xcller and R. Schuhmann, Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) or in the textbook xe2x80x9cIntroduction to Particle Technologyxe2x80x9d by M. Rhodes, Verlag Wiley and Sons (1998) both of which are incorporated herein by reference.